Strep-tag® and One-STrEP-tag
Background
The Strep-tag® was originally selected from a random library for specific streptavidin binding activity thereby enabling purification of corresponding fusion proteins on streptavidin affinity columns. Binding reversibly to the same pocket where the natural ligand D-biotin is complexed, elution of the bound recombinant protein is effected by competition with D-biotin. Analogues with less strong affinity, such as D-desthiobiotin, can be used as well thus facilitating repeated use of the column.
The system was systematically optimized over the years1, including development of the optimized Strep-tag®II for N- or C-terminal as well as protein internal fusion and engineering of a streptavidin variant with improved binding capacity, dubbed Strep-Tactin®.
Properties
A particular benefit of Strep-tag®II is that it has a neutral amino acid composition and that it does not hamper protein folding or secretion, nor does it interfere with protein function. Strep-tag® enables purification of recombinant proteins to over 99% purity in a single step from crude lysates. The extraordinary purification factors are based on i) very low tendency of Strep-Tactin® to bind other proteins non-specifically, ii) highly specifc Strep-tag®II:Strep-Tactin® interaction and iii) specific competitive elution with minute amounts of desthiobiotin in the physiological wash buffer. Moreover, extreme stability of Strep-Tactin® is the basis of robust affinity resins.
Suitability for PPI
Strep-tag® technology was used from the beginning to purify intact protein complexes in a preparative manner, even if just one subunit carries the tag. For example, the cytochrome C oxidase, an integral membrane protein, was isolated after complexation with a cognate recombinant antibody fragment equipped with the Strep-tag® and the crystal structure of the entire assembly was subsequently determined2.
These properties predestine the Strep-tag®:Strep-Tactin® system for PPI analysis according to Rigaut et al., 19993. The development of a tandem arrangement of two Strep-tag®II sequences, dubbed One-STrEP-tag, even improved performance by increasing purification yields of poorly expressed protein complexes and sustaining elevated detergent concentrations to reduce background.
As a conclusion, extremely efficient and fast Strep-tag®II and One-STrEP-tag are reliable tools to meet the challenge of isolating protein complexes at high purity without loosing transient binders.
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